Parkinson's disease (PD) is the second most common neurodegenerative disorder after Alzheimer's dementia. It is estimated that more than one million individuals in the United States of America alone are affected with this disabling disease and more than 50,000 new cases arise each year (Fahn and Przedborski, 2000). A progressive, age-related, neurodegenerative disease characterized by bradykinesia, resting tremor, rigidity and gait disturbance, PD is also characterized by a massive progressive destruction of dopaminergic neurons in the substantia nigra. Like many other neurodegenerative diseases, PD presents itself mainly as a sporadic condition, meaning in the absence of any genetic linkage, but in rare instances, PD can also arise as a simple Mendelian trait, linked to defects in a variety of genes.
Although clinically and pathologically sporadic and familial PD may differ on several significant aspects they all share the same biochemical brain abnormality, namely the dramatic depletion in brain dopamine (Dauer and Przedborski, 2003). The reason why PD patients exhibit low levels of brain dopamine stems from the degeneration of the nigrostriatal dopaminergic pathway, which is comprised of dopaminergic neurons whose cell bodies are located in the substantia nigra and whose projecting axons and nerve terminals are found in the striatum (Vila and Przedborski, 2004).
The best-characterized model of PD has been developed by using the neurotoxin, MPTP (Bloem et al., 1990, Flint Beal, 2001). The discovery of MPTP occurred in 1982 when a group of drug addicts in California developed acute onset of severe Parkinsonism. Investigation revealed that the syndrome was caused by self-administration of a synthetic heroin analogue that had been contaminated by a by-product, MPTP during manufacture. MPTP administration was subsequently shown to model PD in both mice and primates. MPTP is highly lipophilic and it readily crosses the blood-brain barrier. It is then converted into its active metabolite, 1-methyl-4-phenylpyridinium (MPP+) by monoamine oxidase B which is then taken up by high-affinity dopamine and noradrenaline uptake systems and is consequently accumulated within mitochondria of nigrostriatal dopaminergic cells. This can lead to a number of deleterious effects on cellular function, resulting in neuronal cell death (Tatton and Kish, 1997, Tanji et al., 1999). In mice, 2′-CH3-MPTP is a more potent neurotoxic than conventional MPTP showing severe histopathological changes including swelling of cytoplasm, interstitial edema, depletion of dopaminergic neurons with reactive microglial proliferation and gliosis (Abdel-Wahab, 2005).
While most of the research on PD has been conducted with a focus on adults, some reports convincingly demonstrate that systemic MPTP injection into neonatal mice results in permanent brain damage which can be traced in adulthood. The fact that developing brain is vulnerable to MPTP damage as well deserves further investigation.
In addition to MPTP, a number of other chemicals have also been reported to cause neuronal damages in various regions of adult mouse brain. These chemicals include a long list of structurally and functionally diverse compounds, ranging from industrial toxic compounds, agricultural pesticides, to food additives. Once again, most of the neurotoxicity studies conducted thus far have been based on adult models. However it is important to note that the developing brain with less intact blood-brain-barrier is much more vulnerable to damage caused by known neurotoxins, and more critically the damage inflicted cannot be fully and correctly compensated by the developing brain. Therefore the importance of testing chemicals for their potential neurotoxicity in developing brain cannot be over-emphasized, especially in dealing with the issue of silent neurotoxicity (i.e., early exposure to neurotoxicants, but without clinical symptoms until adulthood). This was further exacerbated by the US Environmental Protection Agency's Developmental Neurotoxicity Testing Guideline (DNTG). More recently the European Commission adopted a proposal for a new European Union regulatory framework to test all chemicals through a rigorous regime with estimates of the new measure costing up to seven billion Euros and taking at least ten years to implement.
Glial fibrillary acidic protein (GFAP), an intermediate filament protein expressed predominately in the astrocytes of the central nervous system (CNS), has been proposed by O'Callaghan (O'Callaghan, 1988) to be an early and sensitive biomarker for monitoring neuronal damages in adult rodent brains caused by various neurotoxic agents, including 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (Reinhard et al., 1988, Araki et al., 2001, Chen et al., 2002, Fields and Stevens-Graham, 2002, Kurosaki et al., 2004). An upregulation of GFAP expression has been correlated with increased neurological damage. The traditional methods employed to analyze endogenous GFAP expression include predominantly immunocytochemistry, Northern blot, Western blot and ELISA (O'Callaghan, 1991, Eng et al., 2000).
The GFAP basal promoter consists of a TATA and a CAAT box. Enhancer and silencer sequences are found between −250 and −80 bp and between −1980 and −1500 bp. These positive control regions contain consensus sequences for many transcription factors including a cAMP response element and binding sites for the Sp-1, NF-1, AP-1 and AP-2 transcription factors. Tissue specificity is conferred by a human GFAP consensus sequence located in the −1980 to −1500 bp region.
Reactive gliosis (astrogliosis) occurs in response to almost any insult, physical or chemical, to the central nervous system (CNS) and is characterized by hypertrophy of the astrocyte cell body and its processes, accompanied by an increase in expression of GFAP. Reactive gliosis is accompanied by an up-regulation of GFAP. A similar increase in GFAP occurs following traumatic and toxic injuries to the peripheral nervous system.
With the advancement in mouse transgenics and the availability of novel reporter genes, several reporter genes, including beta-galactosidase (lacZ), green fluorescent protein (GFP), and luciferase (LUC) have been introduced into transgenic mice under the control of the GFAP promoter and proved to be useful surrogates for studying the GFAP transcriptional activity during gliosis in vivo (Brenner et al., 1994, Zhuo et al., 1997, Zhu et al., 2004).
WO 00/02997 and U.S. Pat. No. 6,501,003 describe the generation of a transgenic mouse expressing green fluorescent protein in glial cells. The authors describe the in vitro detection of fluorescence in optic nerve, brain, retina, sciatic nerve and cornea either in whole mounted tissue or sections thereof.